Nucleic acid probes are typically single strands of DNA or RNA that are capable of specifically binding to a complementary nucleic acid. Thus, such probes are used in the biotechnology field to seek out and detect a desired nucleic acid.
Current nucleic acid probe methodology, which may detect a single base mutation under very stringent conditions, typically involves attaching a selected nucleic acid molecule to a nitrocellulose filter by bringing it into contact with the filter directly or via the Southern transfer technique from an agarose gel. The selected nucleic acid molecule is then denatured and the filter is baked to ensure firm attachment. Generally, the preparation of the nucleic acid molecule and the running of the gel is a time-consuming, costly process requiring a reasonably high technical skill level.
The next step is to prepare a probe nucleic acid molecule, by radioactively labeling a specific nucleic acid molecule using procedures well known in the art, such as nick translation or polynucleotide kination. The probe nucleic acid molecule hybridizes with the bound selected nucleic acid molecule at a suitable temperature, typically for several hours. The probe nucleic acid molecule hybridizes with any bound selected nucleic acid molecule that has complementary base sequences. Extraneous material, including unbound probe nucleic acid molecule, is then washed away from the filter and the filter is then exposed to film sensitive to the radioactive label.
In certain instances, the amount of selected nucleic acid probe may be too small for routine hybridization analysis. However, the quantity of a selected nucleic acid molecule that may have a mutation to one or more bases may be increased by subjecting the selected nucleic acid molecule to polymerase chain reaction ("PCR"), as disclosed in U.S. Pat. No. 4,683,195 (Mullis et al.). (Throughout this specification, reference is made to various patents and publications. The disclosures of all such patents and publications in their entireties are expressly incorporated herein by reference.) PCR comprises treating separate complementary strands of the selected nucleic acid molecule with a molar excess of 2 oligonucleotide primers. The primers permit formation of complementary primer extension products, which then act as templates for a next round of synthesizing the selected nucleic acid sequence, thus, the sequence is amplified and may then be detected. A disadvantage of this process is the requirement for thermal cycling, and the potential for carryover contamination due to amplification of the selected nucleic acid molecule, itself. The PCR-amplification products may then be screened with a probe, as described above. In an alternative approach, the PCR-amplification products could be sequenced using traditional methods well known in the art, such as dideoxynucleotide sequence analysis.
In an alternative approach, U.S. Pat. Nos. 4,876,187 and 5,011,769 (both to Duck et al.) disclose nucleotide sequences having scissile linkages that are useful for the detection of selected nucleic acid sequences. However, neither patent is specifically directed to the detection of a single nucleotide mismatch between a desired sequence and other sequences present in a sample.